Human agmatinase is an enzyme that plays a role in the hydrolysis of agmatine [4-(aminobutyl)guanidine] to putrescine and other polyamines such as spermine and spermidine which are essential for DNA replication, cell homeostasis and cell transformation. Polyamines are required for entry and progression of the cell cycle. Also, augmentation of polyamine levels is essential for cellular transformation. Agmatine is a metabolite of arginine via arginine decarboxylase (ADC) and is implicated in the attenuation of cellular polyamine levels (Satriano et al. (1998) J. Biol. Chem. 273 (25):15313-15316). Agmatine represents an alternate pathway to polyamine production in contradistinction to the well-studied pathway mediated by ornithine decarboxylase (ODC) which acts in the metabolism of arginine to yield putrescine which feeds into polyamine synthesis. End-products of arginine metabolism include the cell-signaling molecules: NO, glutamate, and agmatine. (Wu et al., (1998) Biochem. J. 336:1-17). Mammalian ADC is membrane associated and expressed in the inner membranes of mitochondria.
Agmatine is widely and unevenly distributed in a variety of mammalian tissues including serum. The tissues where agmatine has been identified include: stomach, aorta, small intestine, large intestine, spleen, lung, vas deferens, adrenal gland, kidney, heart, liver, skeletal muscle, testes, and brain. The highest concentration was found in stomach, aorta, and small intestine (Raasch et al., (1995) Life Sciences 56 pp. 2319-2330). Agmatine binds to α2-adrenergic and imidazoline receptors and is bioactive in a number of tissues (Wu et al., (1998) Biochem. J. 336:1-17). It is contained in neurons and is found in serum which is consistent with its role as a putative neurotransmitter and/or a hormone. Agmatine potentiates the analgesic effects of morphine and clonidine in a dose-dependent manner and decreases the EC50 of morphine and clonidine by more than 75% in a mouse tail-flick test. Intrathecal agmatine at high doses causes a decrease in the pain threshold (Jin, Li et al., (1999) Acta Pharmacologica Sinica 20 (1): 81-85).
The enzyme has been isolated from rat brain and was localized primarily in the mitochondria wherein it degrades the substrate, agmatine at its site of action (Regunathan et al.,(1996) J. Neurochem. 67(4):1761-65). Specifically, the enzyme is localized in the mitochondrial matrix.
It is presumed that agmatine is a biologically active molecule with numerous physiological roles including, but not limited to: binding to α2-adrenergic and imidazoline receptors, causing release of catecholamine from adrenal chromaffin cells, stimulating release of insulin and uptake of Ca in pancreatic cells, inhibitor of lipolysis in rat adipocytes, increase glucose uptake and glycogen content of the rat diaphragm, and increase glucose oxidation and lipogenesis in fat pads and glucose oxidation in isolated fat cells (Raasch et al., (1995) Life Sciences 56 pp. 2319-2330).
The concentration of agmatine in the whole brain is comparable to that of other neurotransmitters. It is unevenly distributed with the highest concentration in the hypothalamus, forebrain, and cerebral cortex (Reis et al. (1999) Annals of the NY Academy of Sciences 881: 65-80). Agmatine is synthesized and stored in astrocytes (Youngson et al., Ann. NY Acad. Sci. 763: 440-444).
Agmatinase (agmatine ureohydrolase) is an enzyme that hydrolyzes agmatine to form putrescine and urea. Putrescine along with spermine and spermidine are polyamines.
Polyamines such as putrescine, spermidine, and spermine are required for DNA replication, proliferation, and cell homeostasis. Ornithine decarboxylase (ODC) is the first rate-limiting enzyme of polyamine biosynthesis and one of the most highly regulated eukaryotic enzymes. Cellular polyamine transporters are stimulated by many of the same factors that induce ODC activity. Cellular polyamine uptake occurs both in normal and rapidly proliferating cells and tumor lines (Moulinoux et al. (1991) Cell. Mol. Biol. 37:773-783; Bogle et al. (1994) Am. J. Physiol. 266:C776-C783; Holley et al. (1992) Cancer Res. 52:4190-4195).
Polyamines have been reported in the herpes simplex virion (HSV) (Gibson et al. (1973) Polyamines in Normal and Neoplastic Growth, edited by D. H. Russell, Raven Press, NY). The polyamines may serve as specific structural components of the virion and serve to neutralize the electronegativity of DNA. Agmatinase may play a role in HSV infection as it is induced during the latent phase of HSV replication (unpublished data). Also, 25312 expression is induced during infection by the DNA virus HBV (unpublished data). Thus, high levels of polyamine synthesis via the agmatinase pathway may be a requirement for DNA viruses.
Intracellular polyamine concentrations are autoregulated by the induction of the protein antizyme (Matsufuji et al. (1995) Cell 80:51-60). Antizyme binds to ODC and inhibits its activity and accelerates its degradation (Hayashi et al. (1996) Trends Biochem Sci. 21:27-30). More recently, antizyme has been shown to suppress polyamine transporters (Mitchell et al. (1994) Biochem. J. 299:19-22; Suzuki (1994) Proc. Natl. Acad. Sci. 91:8930-8934). Thus, antizyme through its ability to suppress both the polyamine biosynthetic enzyme ODC and polyamine transporters is an effective endogenous mechanism for limiting intracellular polyamine levels.
Recent research has demonstrated the induction of antizyme by agmatine. The induced antizyme can bind to ornithine decarboxylase (ODC) and depress polyamine biosynthesis and transport. (Satriano et al. (1998) J. Biol. Chem. 273: 15313-15316). The capacity of agmatine to induce antizyme is demonstrated by (a) an agmatine-dependent translational frameshift of antizyme mRNA to produce a full-length protein and (b) suppression of agmatine-dependent inhibitory activity by either anti-antizyme IgG or antizyme inhibitor (Satriano et al. (1998) J. Biol. Chem. 273 (25):15313-15316).
There is evidence that agmatine has several potential roles in mammalian physiology, including: acting as a neurotransmitter, as a secretogogue and as an endogenous inhibitor of all isoforms of NOS, and it may play a role in modulating the state of macrophage activation during inflammation by regulating NOS activity and NO production (Sastre et al. (1998) Biochem. J. 330:1405-1409).
Agmatine (AGM) has long been characterized as a constituent of bacteria, plants and some invertebrates (Tabor and Tabor (1984) Ann. Rev. Biochem. 53:749-790). More recently, agmatine was shown to be expressed in rat brain (Li, G. et al. (1994) Science 263:966-969). Agamatine is an endogenous ligand at imidazoline and α-adrenergic receptors to which it binds with high affinity (Tabor et al. (1984) Ann. Rev. Biochem. 53:749-790). Agmatine also has properties of an endogenous neurotransmitter. However, its actual role in normal brain function has not yet been established (Reis et al. (1998) Adv Pharmacol 42:645-9). Agmatine is locally synthesized in the brain and stored in a large number of neurons with selective distribution in the central nervous system. Also, it can be enzymatically degraded by agmatinase in the synaptosomes (Reis et al., (1999) Annals of the NY Academy of Sciences 881:65-80.)
Agmatine which is an endogenous ligand of imidazoline receptors is biologically active in the nervous system and many other tissues in mammals (Li et al., (1994) Science, 263:966-969).
Agmatine stimulates the release of catecholamines from adrenal chromaffin cells, increases arterial blood pressure when injected into rats, stimulates the release of insulin from β-cells in pancreatic islets, and increases the release of gonadorelin from the hypothalamus (Galea et al., (1996) Biochem. J. 316:247-249). Also, it potentiates opioid analgesia and prevents the tolerance induced by opioids (Kolesnikov et al., (1996) Eur. J. Pharmacol. 296:17-22). Agmatine has analgesic effects and potentiates morphine and clonidine analgesia by activation of imidazoline receptors, but cannot prolong the analgesic time of morphine (Li, et al., (1999) Acta Pharmalogica Sinica 20(1):81-85). Therefore, regulation of agmatine degradation may be useful in the treatment of pain.
Agmatine is an antimitogen capable of inhibiting the proliferation of vascular muscle cells (Reis et al., (1999) Annals of the NY Academy of Sciences 881:65-80.)
Agmatine has been shown to play a role in modulating the state of macrophage activation during inflammation (Sastre et al., (1998) Biochem. J. 330:1405-1409). Sastre et al. demonstrated that macrophages express the enzymes ADC and agmatinase and that the enzyme activities are regulated during inflammation. ADC and agmatinase are constituitively expressed in macrophages and that lipopolysaccharides (LPS) dose-dependently and reversibly modulated the basal and evoked activity of both enzymes as well as initiating induction of iNOS indicating that the enzymes are regulated. Agmatine is an inhibitor of all isoforms of nitric oxide synthases (NOS) (Reis et al. (1999) Annals of NY Academy of Science 881: 65-80).
Agmatine can play a role in the etiology of viral infections, specifically Herpes Simplex Virus (HSV) as relates to the formation of polyamines in the virus (Gibson and Roizman, (1973) Polyamines in Normal and Neoplastic Growth. ed. Russell, Raven Press, NY). The polyamines, which can be produced due to the action of agmatinase on agmatine, can act as specific structural components of the virion. It was demonstrated that highly purified preparations of enveloped HSV contain the polyamines spermidine and spermine in a nearly constant molar ratio of 1.6±0.2 (Gibson and Roizman (1971) Proc. Nat. Acad. Sci. U.S.A. 68:2818-2821). Thus, the polyamines could serve to neutralize the electronegativity of the DNA.
The polyamines have been compartmentalized to the nucleocapsid in the HSV. Moreover, there is a segregation of the spermine and spermidine in the HSV with spermine inside the nucleus and spermidine outside (Gibson and Roizman (1971) Proc. Nat. Acad. Sci. USA 68: 2818-2821).
It has been observed that agmatinase activity is expressed with regional variability in the rat. The highest levels were observed in the hypothalamus, moderate expression in the medulla oblongata and hippocampus and lowest levels in the striatum and cerebral cortex (Sastre et al. (1996) J. Neurochem. 67: 1761-65).
Carvajal et al. (1999) Biochem. Biophys. Res. Comm. 264:196-200 discloses a coupled urease system that can be used to assay agmatinase activity and binding. Agmatinase activity can also be assayed using a two-step procedure in which [guanido-14C] agmatine is first hydrolyzed to [14C] urea and putrescine and then [14C] urea is hydrolyzed by added urease to 14CO2 and NH3. This method has been widely used to assay bacterial agmatinases (Satishchandran C. et al. (1986) J. Bacteriol. 165:843-848). This method can be adapted for use in mammalian tissues (Sastre et al. (1996) J. Neurochem. 67: 1761-65). It has been determined that the agmatinase isolated from rat brain has maximal activity a pH 8-8.5 and an apparent Km of 5.3±0.99 mM. There are some known inhibitors of agmatinase including the divalent cation Mn+2 (Sastre et al., (1996) J. Neurochem 67: 1761-1765) and other organic inhibitors including N-isoamylene agmatine (Khramov.V. (1976) Vopr Med Khim 22(6): 804-808). and the organic inactivator diethyl pyrocarbonate (DEPC) (Carvajal, N. et al. (1999) Biochem. Biophys. Res. Commun. 264 (1):196-200).
Mechanistic studies have been performed with agmatinases isolated from bacteria such as E. coli to ascertain the critical sites in the native protein for catalytic function and substrate binding (Carvajal (1999) Biochem. Biophys. Res. Commun. 264(1):196-200). Additionally, various inhibitors have been identified which affect agmatinase activity, including N-isoamylene agmatine (Kharamov (1976) Vopr Med Khim 1976 22 (6):804-808). Additionally, ornithine and arginine have been shown to be inhibitors of agmatinase. Ornithine inhibited agmatinase in E.coli noncompetitively while it inhibited arginine competitively (Satischandran et al. (1986) J. Bacteriol. 165: 843-848). EDTA and EGTA were shown to be irreversible inactivators of agmatinase. In the bacterium, E.coli, studies indicated that agmatinase requires a metal for its structural stability rather than its catalytic activity, and that its production is induced by agmatine and that it serves a role in the production of the polyamine putrescine and that it is not a major source of carbon and energy (Satischandran et al. (1986) J. Bacteriol. 165: 843-848).
The reaction catalyzed by agmatinase is similar to that catalyzed by arginase (EC 3.5.3.1) which hydrolyzes arginine to ornithine and urea. However, in rat brain differences have been noted. Arginase activity localized primarily in the soluble and cystolic fractions, whereas agmatinase localized primarily in the mitochondria. (Sastre, et al. (1996) J. Neurochem. 67: 1761-1765).
Thus, agmatine, as a potential metabolic precursor for polyamines, plays a key role in cellular physiology and cell homeostasis. It can have several important biochemical effects ranging from but not limited to effects on the central nervous system, the cardiovascular system, inflammation, pain analgesia, cell proliferation in cancer, and viral replication.
Accordingly, agmatinases are a major target for drug action and development. Thus, it is valuable to the field of pharmaceutical development to identify and characterize novel agmatinases and tissues and disorders in which agmatinases are differentially expressed. The present invention advances the state of the art by providing a novel human agmatinase and tissues and disorders in which expression of a human agmatinase is relevant. Accordingly, the invention provides methods directed to expression of the agmatinase.